Bisecting-GlcNAc on Asn388 is characteristic to ERC/mesothelin expressed on epithelioid mesothelioma cells

Abstract

Mesothelioma is a highly aggressive tumour associated with asbestos exposure and is histologically classified into three types: epithelioid-type, sarcomatoid-type and biphasic-type. The prognosis of mesothelioma patients is poor and there is no effective molecular-targeting therapy as yet. ERC/mesothelin is a glycoprotein that is highly expressed on several types of cancers including epithelioid mesothelioma, but also expressed on normal mesothelial cells. This is a predicted reason why there is no clinically approved therapeutic antibody targeting ERC/mesothelin. In the present study, we focussed on the differential glycosylation between ERC/mesothelin present on epithelioid mesothelioma and that on normal mesothelial cells and aimed to reveal a distinct feature of epithelioid mesothelioma cells. Lectin microarray analysis of ERC/mesothelin using cells and patient specimens showed significantly stronger binding of PHA-E4 lectin, which recognizes complex-type N-glycans having a so-called bisecting-GlcNAc structure, to ERC/mesothelin from epithelioid mesothelioma cells than that from normal mesothelial cells. Further, liquid chromatography/mass spectrometry analysis on ERC/mesothelin from epithelioid mesothelioma cells confirmed the presence of a bisecting-GlcNAc attached to Asn388 of ERC/mesothelin. These results suggest that this glycoproteome could serve as a potential target for the generation of a highly selective and safe therapeutic antibody for epithelioid mesothelioma.

Keywords: ERC/mesothelin; bisecting-GlcNAc; epithelioid mesothelioma; glycosylation.

Graphical abstract

Fig. 1.

PHA-E4 lectin strongly binds to ERC/MSLN from epithelioid mesothelioma cells compared to that from normal mesothelial cells. (A) Schematic representation of ERC/MSLN. Asn388, Asn496 and Asn523 indicate glycosylation sites. (B) Western blotting to confirm the immunoprecipitation of ERC/MSLN from cell lines. Arrow indicates ERC/MSLN. Elute x 10: 10 times amount of elutes were loaded. (C–F) Scan images and bar graphs of glycan profiling analysis of ERC/MSLN from MeT-5A (C), MES-F (D), MESO-4 (E) and H226 cells (F) using lectin microarray (n = 3). White squares in scan images and arrows in bar graphs indicate the position of PHA-E₄ lectin. The numbers above the arrows indicate the net intensity of PHA-E₄ lectin. Error bar indicates standard deviation.

Fig. 2.

PHA-E4 lectin binds to N-glycans on ERC/MSLN from epithelioid mesothelioma cell lines and to ERC/MSLN from epithelioid mesothelioma patient specimens to a similar extent. (A) Western blotting using anti-ERC/MSLN antibody to validate PHA-E₄ binding against ERC/MSLN. Lower panel shows the amount of IP-ERC/MSLN used for lectin (PHA-E₄) precipitation (input). Upper panel shows the amount of precipitated IP-ERC/MSLN. Arrow indicates ERC/MSLN. (B) Scan image and bar graph of glycan profiling of PNGase F-treated ERC/MSLN from H226 cells. (C) Scan image and bar graph of glycan profiling of ERC/MSLN from patient specimens (n = 3). A representative data among four patient specimens is shown (C). White squares in scan images and arrows in bar graphs indicate the position of PHA-E₄ lectin. The numbers above the arrows indicate the net intensity of PHA-E₄ lectin. Error bar: standard deviation. (D) Comparison of relative PHA-E₄ binding intensity among cell lines (MeT-5A, MES-F, MESO-4, H226) and patient specimens (patients 1–4). Error bar indicates standard deviation. Wilcoxon rank sum test was used for statistical analysis. ^***P < 0.005, n.s., not significant.

Fig. 3.

Bisecting-GlcNAc, the glycan structure recognized by PHA-E4, predominantly exists on the Asn388 residue of ERC/MSLN derived from epithelioid mesothelioma cells. (A) Silver staining result of IP-ERC/MSLN from H226 cells. Dotted line square indicates ERC/MSLN and digested position used for LC/MS analysis. (B) Flow chart showing the sample preparation of ERC/MSLN from H226 cells for glycan analysis by LC/MS. (C–E) Glycan profile of ERC/MSLN at Asn388 (C), Asn496 (D) and Asn523 (E). Black bar indicates glycan structures containing bisecting-GlcNAc, and white bar indicates glycan structures without bisecting-GlcNAc. (F) Expected glycan structure containing bisecting-GlcNAc. dHex, deoxyhexose; Hex, hexose; HexNAc, N-acetylhexosamine; NeuAc, N-acetylneuraminic acid.